In this study, the feasibility of molecular depth profiling using dual beam sputtering of biological materials is examined. The model system is a 402-nm Langmuir Blodgett multilayer film consisting of 149 monolayers of barium arachidate (AA). The thin film was initially subjected to pre-bombardment with a 15-keV Au+ beam. Subsequently, an imaging depth profile experiment was performed on the pre-irradiated sample using a 40-keV C60+ beam. An extremely low erosion rate under Au+ bombardment is found on this model system. In the subsequent C60+ depth profiles, surprisingly large molecular ion signals are detected at the gold pre-irradiated surface. These signals then rapidly decay to nearly zero, indicating a damaged sub-surface layer being generated by the Au+ pre-bombardment. The thickness of the damaged layer is found to increase with increasing gold ion fluence and saturate at about 100 nm at 6 × 1014 Au+/cm2. This altered layer thickness is significantly larger than the value of ~50 nm obtained on a trehalose film pre-bombarded with a Ga+ source. The results also show that the damage caused by the Au+ beam can be removed by C60+ sputtering and that the molecular information is restored after damage removal. Copyright © 2012 John Wiley & Sons, Ltd.